CN115381954A

CN115381954A - Combined medicine for treating osteosarcoma

Info

Publication number: CN115381954A
Application number: CN202110560718.6A
Authority: CN
Inventors: 赵子建; 许丽君; 周素瑾; 李芳红; 赵正刚
Original assignee: Guangzhou Huazhen Pharmaceutical Co ltd
Current assignee: Shenzhen Hanhui Pharmaceutical Technology Co ltd
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2022-11-25
Anticipated expiration: 2041-05-21
Also published as: CN115381954B

Abstract

The invention provides a combination medicament for the treatment of malignant tumours, in particular osteosarcoma, which comprises the PDE4 inhibitor ZL-n-91 and chloroquine phosphate. The combined medicine can effectively treat osteosarcoma, has the effect obviously superior to that of a PDE4 inhibitor ZL-n-91 and chloroquine phosphate which are used independently, shows that the two have obvious synergistic effect after being used in a matched mode, has strong anti-tumor activity and small side effect, and provides a new treatment option for patients who lose clinical operation indications, cannot perform operations and have poor radiotherapy and chemotherapy effects.

Description

Combined medicine for treating osteosarcoma

Technical Field

The application belongs to the field of antitumor drugs, and particularly provides a combined drug for treating osteosarcoma, which contains a PDE4 inhibitor ZL-n-91 and chloroquine phosphate.

Background

Osteosarcoma (OS) is one of the most common malignant bone tumors, defined by the World Health Organization (WHO) as a primary malignant bone tumor consisting of mesenchymal cells producing osteoid and immature bone. Osteosarcoma often occurs in active growth sites, such as the lower femur, the upper tibia, the upper humerus, etc., and its clinical manifestations include: bone or joint pain, localized swelling, lameness. When the disease condition further worsens to the general state, it manifests as fever, malaise, weight loss, anemia and failure. In most countries, the age-normalized ratio (ASR) of osteosarcoma is 0.20-0.35/10 ten thousand, accounting for 20-40% of bone tumors, wherein in china it accounts for 20%. Osteosarcoma occurs in adolescents at a high rate in men compared to women, and the overall survival rate of patients with osteosarcoma has changed little, approximately 70%, over the last thirty years. But for patients with metastatic disease and those who do not respond well to initial therapy, 5-year survival rates are only 25% in people between 2 and 68 years of age. Therefore, the search for new molecular targets is of great significance for the early diagnosis and targeted therapy of osteosarcoma patients. The current treatment modes of osteosarcoma are new auxiliary chemotherapy before operation, surgical resection and auxiliary chemotherapy after operation. Chemotherapy drugs include Methotrexate (MTX), adriamycin (ADM), cisplatin (DDP), and Ifosfamide (IFO), but high doses of chemotherapy drugs cause significant side effects, and can cause rare toxic effects such as cardiotoxicity, acute and chronic nephrotoxicity, neurotoxicity, hearing loss, and infertility, in addition to common complications such as alopecia, myelosuppression, mucositis, nausea, and vomiting. Therefore, further research is necessary to identify effective drugs and develop new therapeutic strategies with less side effects to treat this fatal disease.

Research shows that the phosphodiesterase 4 inhibitor (PDE 4 i) Roflumilast (Roflumilast) can obviously inhibit the growth of ovarian cancer, lung cancer and B cell lymphoma; in another study, PDE4i Rolipram (Rolipram) was found to inhibit proliferation of the human mandibular OS cell line HOSM-1, suggesting that PDE4i has potential anti-OS effects. Rolipram is the first prototype PDE4 inhibitor but fails to market first due to its severe adverse effects and is commonly used as a positive control in subsequent studies of PDE4 inhibitors. In addition to Rolipram, researchers have further developed a new generation of PDE4 inhibitors, of which Roflumilast, apremilast, and Crisborole were continuously approved for marketing. Oral Roflumilast was approved in the european union and us in 2010 and 2011 for the treatment of severe COPD and asthma symptoms, apremilast was approved in the us (2014) for active psoriatic arthritis patients and moderate to severe plaque psoriasis patients, and crisabarole was approved in the us (2016) for topical treatment of mild to moderate atopic dermatitis in patients 2 years and older. However, the PDE4 inhibitors have a narrow therapeutic window and are associated with side effects such as nausea and vomiting during treatment. Several novel PDE4 selective inhibitors have been developed for the above side effects, such as ZL-n-91, developed by Kyohimu, university of North Carolina:

ZL-n-91 is a novel selective PDE4i, which is several thousand times more selective than other PDE family molecules. The 2008 Ruihong Ma research shows that ZL-n-91 has an inhibition effect on IL-17production by memory T helper (Th) 17 cells, and has no influence on cell viability. A2010 study by Hui-Fang Tang et al found ZL-n-91 to improve the smoking (CS) -induced inflammatory response in a COPD-like model in male Sprague-Dawley rats, and the authors further found ZL-n-91 to be useful in the treatment of lipopolysaccharide-induced Acute Lung Injury (ALI) in mice. In addition, the study finds that ZL-n-91 can inhibit the proliferation of prostate cancer cell PC-3, but no actual study verification exists for the curative effect of other cancers.

Chloroquine phosphate, chloroquine diphosphate salt, CAS number 50-63-5, structural formula:

the chloroquine is a classical antimalarial drug, and researches in recent years show that the chloroquine has antitumor activity and has various action mechanisms on tumors, such as tumor cell apoptosis induction, autophagy inhibition and the like. The chloroquine phosphate serving as a weakly alkaline medicine can selectively enter acidic lysosomes of tumor cells, effectively inhibit the acidic lysosomes of the tumor cells to inhibit autophagy and the paradoxical reaction _p The action of H normal cells is weak, which lays a good microenvironment foundation for chloroquine to better act on tumor cells and reduce adverse reactions of normal tissues. Research shows that autophagy can reduce the sensitivity of chemotherapeutic drugs by regulating energy metabolism in a stress environment and promote the survival of tumor cells. The autophagy inhibitor chloroquine can effectively prevent autophagy in the process of tumor toxicity chemotherapy, and the blocking of autophagy can promote tumor cells to die and increase the sensitivity of the tumor cells to chemotherapeutic drugs. Thus, targeted drugs and regulated autophagy bidirectional interventions could be an effective cancer treatment strategy.

Disclosure of Invention

The invention aims to provide a combined medicine for treating osteosarcoma, which solves the problem that the medicine effect is influenced due to the fact that the body is intolerant caused by the fact that the PDE4 inhibitor ZL-n-91 is singly used for treating high concentration. The invention uses in vitro tumor cell culture, and researches the combined effect of ZL-n-91 and chloroquine through cell proliferation experiments, plate clone formation experiments and apoptosis experiments. The experiment proves that: chloroquine phosphate (CQ) has synergistic effect on ZL-n-91 in inhibiting proliferation of MNNG/HOS, U2OS and Saos-2 in osteosarcoma cells, and can enhance the apoptosis promoting effect mediated by ZL-n-91.

In one aspect, the present application provides a combination comprising a PDE4 inhibitor and chloroquine phosphate for the treatment of a malignant tumor.

In another aspect, the application provides the use of a PDE4 inhibitor and chloroquine phosphate in the manufacture of a combination for the treatment of a malignant tumor.

Further, the malignant tumor is osteosarcoma.

Further, the PDE4 inhibitor is ZL-n-91.

Further, the drug inhibits tumor cell proliferation.

Further, the drug induces apoptosis of tumor cells.

Further, the molar ratio of the PDE4 inhibitor to the chloroquine phosphate in the medicament is 40.

Further, the molar ratio of the PDE4 inhibitor to the chloroquine phosphate in the medicament is 20.

Further, the molar ratio of the PDE4 inhibitor to chloroquine phosphate in the medicament is 10.

Further, the PDE4 inhibitor and the chloroquine phosphate in the medicament are administered simultaneously or separately.

Further, the medicine is in an injection form.

The chloroquine phosphate described in the present application may be used interchangeably with chloroquine, chloroquine phosphate, etc.

PDE4 inhibitors described herein include, but are not limited to, known and studied PDE4 inhibitors such as theophylline, rolipram, roflumilast, cilomilast, aplet, ZL-n-91, and the like

Osteosarcomas described herein include various types of osteosarcomas of various characteristics, including, but not limited to, traditional osteosarcoma, intramedullary well-differentiated osteosarcoma, paraosteosarcoma, periosteal osteosarcoma, capillary-expanded osteosarcoma, and small cell osteosarcoma.

The formulation and specification of ZL-n-91 and chloroquine phosphate herein may be the same or different.

The ZL-n-91 and chloroquine phosphate in the application are preferably in an injection form, and can also be in an oral preparation form, and the specific preparation form can be an oral preparation form or an injection form, and comprises but is not limited to tablets, capsules, oral liquid preparations, pills, granules, powder, water injection, oil injection, emulsion injection, powder injection and the like.

In addition to oral or injectable dosage forms, other known or under-developed dosage forms such as transdermal administration, inhalation administration, targeted carrier administration, may be routinely selected and designed by those skilled in the pharmaceutical arts as appropriate.

In the treatment of osteosarcoma, ZL-n-91 may be used in combination with other known or developed agents for osteosarcoma treatment or formulated into the same formulation, including but not limited to chemotherapeutic agents and targeted agents.

The phosphodiesterase 4 inhibitor ZL-n-91 of the present invention can be purchased directly, synthesized by itself or by a person with reference to the existing literature, for example, ruihong Ma, bin-yan Yang, chang-you Wu. A selective phosphodiesterase 4 (PDE 4) inhibitor ZL-n-91 primers IL-17reduction by human memory 17 cells, international immunopharmacology,2008,8 (10): 1408-1417.

Has the advantages that: the selective PDE4 inhibitor ZL-n-91 can obviously inhibit the proliferation of tumor cells, and is expected to become an important target for anti-osteosarcoma proliferation research; meanwhile, chloroquine phosphate has the effect of inhibiting autophagy, can enhance the killing effect of ZL-n-91 on osteosarcoma cells, and has good application prospect on treatment of osteosarcoma.

Drawings

FIG. 1 is a graph showing the effect of ZL-n-91 in combination with CQ on the proliferation inhibition of osteosarcoma cells MNNG/HOS, saos-2, U2 OS: setting CQ concentration as 10 μ M, setting ZL-n-91 concentration as 200 μ M, treating the single drug group for 48h, pretreating the combined group with CQ for 2h, treating with ZL-n-91 for 48h, and detecting cell proliferation activity by viable cell counting method. All data are expressed as mean ± standard deviation. (n = 3), P <0.05, P <0.01, P <0.001.

FIG. 2 is a graph showing the effect of ZL-n-91 in combination with CQ on the interaction of MNNG/HOS, saos-2, U2OS osteosarcoma cells: the concentrations of CQ in single group were set to 0,2.5,5,10,20. Mu.M, that of ZL-n-91 in single group to 0,25,50,100,200. Mu.M, and the ratio of drug concentrations in combination group to 1 was set to 10, and 4 drug concentration groups were set, i.e., group one (2.5. Mu.M CQ + 25. Mu.M ZL-n-91), group two (5. Mu.M CQ + 50. Mu.M ZL-n-91), group three (10. Mu.M CQ + 100. Mu.M CQ-n-91), and group four (20. Mu.M CQ + 200. Mu.MZL-n-91), and the proliferation activities of various cells were determined by a viable cell count method, respectively by pre-treating MNNG/HOS, saos-2 and U2OS for 2h and then with ZL-n-91 for 48h. CalcuSyn software was used to analyze the CI values for CQ in combination with ZL-n-91. When the CI values are all less than 1, the synergistic effect is achieved, when the CI is equal to 1, the additive effect is achieved, and when the CI is greater than 1, the antagonistic effect is achieved.

FIG. 3 is a graph showing the effect of ZL-n-91 in combination with CQ on the proliferation inhibition of osteosarcoma cells MNNG/HOS, saos-2, U2 OS: cells in logarithmic growth phase are inoculated into a six-hole plate at proper concentration, and after the cells adhere to the wall, the drugs are added for treatment. Setting CQ concentration at 10 μ M, setting ZL-n-91 concentration at 200 μ M, treating the single drug group for 48h, pretreating the combined group with CQ for 2h, treating with ZL-n-91 for 48h, and changing fresh culture medium. The culture was terminated when macroscopic cell clones appeared, and the number of cell clones formed was counted by fixed staining observation. (n = 3), P <0.05, P <0.01, P <0.001.

FIG. 4 is a graph showing the pro-apoptotic effects of ZL-n-91 in combination with CQ on osteosarcoma cells MNNG/HOS, saos-2, U2 OS: setting CQ concentration as 10 μ M, setting ZL-n-91 concentration as 200 μ M, treating the single drug group for 48h, pretreating the combined group with CQ for 2h, treating with ZL-n-91 for 48h, and detecting apoptosis by flow cytometry. All data are expressed as mean ± standard deviation. (n = 3), P <0.05, P <0.01, P <0.001.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

Example 1

Living cell counting method for detecting influence of combination of ZL-n-91 and CQ on osteosarcoma cell proliferation

1) Single cell suspensions were prepared from logarithmic phase cells U2OS, saos-2, MNNG/HOS. The cells were inoculated into six-well plates at 2ml per well, and divided into 4 groups: solvent control group (added with equal volume of solvent EtOH), CQ group (10. Mu.M), ZL-n-91 group (200. Mu.M), CQ + ZL-n-91 group, each group of 3 secondary wells;

2) After the cells adhere to the wall, adding CQ for pretreatment for 2 hours, and then adding ZL-n-91 for treatment for 48 hours;

3) After removing old medium, washing twice with PBS, cells were collected by trypsinization and counted.

The results are shown in FIG. 1: the CQ and ZL-n-91 can inhibit the proliferation of MNNG/HOS, U2OS and Saos-2 cells of osteosarcoma cells, and the cell activity of the combination of the CQ and the ZL-n-91 is further reduced.

Example 2

Calculation of CQ and ZL-n-91 combined action index

1) Single cell suspensions were prepared from cells in logarithmic growth phase U2OS, saos-2, MNNG/HOS. Inoculating 2ml of cell suspension per well into a six-well plate, setting 3 groups (CQ group alone, ZL-n-91 group alone, and CQ + ZL-n-91 group) for each cell, and 3 auxiliary wells for each group;

2) CQ working concentration is set to be 0,2.5,5,10 and 20 mu M, ZL-n-91 single group concentration is set to be 0,25,50,100 and 200 mu M, and the concentration ratio of the medicines in the combined group is 1.

3) After the cells adhere to the wall, the combined group is pretreated for 2 hours by CQ and then treated for 48 hours by ZL-n-91.

4) After removing old culture medium and washing with PBS twice, the cells are collected by trypsinization and counted to detect the cell viability. Data were entered into CalcuSyn software to calculate the Combination Index (CI). Wherein the CI value is less than 1, the synergistic effect is obtained, the additive effect is obtained when the CI value is equal to 1, and the antagonistic effect is obtained when the CI value is greater than 1.

The results are shown in FIG. 2: CQ has the capability of inhibiting osteosarcoma cell proliferation in cooperation with ZL-n-91

Experimental example 3

Plate clone formation experiment for detecting influence of ZL-n-91 and CQ on osteosarcoma cell clone formation capability

1) Single cell suspensions were prepared from cells in logarithmic growth phase U2OS, saos-2, MNNG/HOS. The cells were inoculated in six well plates at 2ml per well, and divided into 4 groups: solvent control group (added with equal volume of solvent EtOH), CQ group (10. Mu.M), ZL-n-91 group (200. Mu.M), CQ + ZL-n-91 group, each group of 3 secondary wells;

2) After the cells adhere to the wall, adding CQ for pretreatment for 2h, then adding ZL-n-91 for treatment for 48h, and replacing with fresh culture medium;

3) And (3) terminating the culture after 10 days of culture when macroscopic cell clones appear, fixing the cells by using a paraformaldehyde solution, carrying out crystal violet staining, washing by using PBS (phosphate buffer solution), marking more than or equal to 50 cell clone groups as 1 cell clone, observing and counting the formation number of each group of cell clones, and expressing the result by the formation number of the cell clones.

The results are shown in FIG. 3: CQ can remarkably enhance the inhibition effect of ZL-n-91 on the clonogenic capacity of osteosarcoma cells.

Experimental example 4

Flow cytometry detection of induction effect of combination of ZL-n-91 and CQ on osteosarcoma apoptosis

1) Single cell suspensions were prepared from logarithmic phase cells U2OS, saos-2, MNNG/HOS. The cells were inoculated in six well plates at 2ml per well, and divided into 4 groups: solvent control group (adding equal volume of solvent EtOH), CQ group (10. Mu.M), ZL-n-91 group (200. Mu.M), CQ + ZL-n-91 group, each group with 3 auxiliary holes;

3) After 48h cells were harvested, washed 2 times with cold PBS, 100. Mu.l of 1 BindingBuffer was added to prepare a cell suspension in a flow tube, 5. Mu.l of 7AA-D and 5. Mu.L of PE were added according to the kit instructions for staining, gently vortexed cells, incubated for 15min at room temperature in the dark, 200u1 BindingBuffer was added to the tube, flow cytometry was performed over 1h, and the results were analyzed by FlowJoV10 analytical software.

The results are shown in FIG. 4: CQ can remarkably enhance the apoptosis promoting effect mediated by ZL-n-91.

The research results show that the combined action of the phosphodiesterase 4 inhibitor ZL-n-91 and CQ can inhibit the cell proliferation of osteosarcoma, and the effect is superior to that of the single use of CQ and ZL-n-91, and the anti-tumor effect is good.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make modifications and alterations to the present invention in light of the present disclosure.

Claims

1. A combination for the treatment of a malignant tumour comprising a PDE4 inhibitor and chloroquine phosphate.

Use of a pde4 inhibitor and chloroquine phosphate in the preparation of a combination medicament for the treatment of malignant tumours.

3. The combination according to claim 1 or the use according to claim 2, wherein the malignancy is osteosarcoma.

4. The combination or use according to claim 3, wherein the malignant tumor is a traditional osteosarcoma, an intramedullary well-differentiated osteosarcoma, a paraosteosarcoma, an periosteal osteosarcoma, a capillary expanded osteosarcoma, a small cell osteosarcoma.

5. The combination according to claim 1 or the use according to claim 2, wherein the PDE4 inhibitor is selected from the group consisting of theophylline, rolipram, roflumilast, cilomilast, aplidine, ZL-n-91.

6. The combination or use according to claim 4, wherein the PDE4 inhibitor is ZL-n-91.

7. The combination according to claim 1 or the use according to claim 2, wherein the medicament inhibits tumor cell proliferation.

8. The combination as claimed in claim 1 or the use as claimed in claim 2, wherein the medicament induces apoptosis of tumour cells.

9. The combination according to claim 1 or the use according to claim 2, wherein the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is from 40.

10. The combination or use according to claim 9, wherein the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is 20.

11. The combination or use according to claim 10, wherein the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is 10.

12. The combination according to claim 1 or the use according to claim 2, wherein the PDE4 inhibitor and chloroquine phosphate in the medicament are administered simultaneously or separately.

13. A combination as claimed in claim 1 or use as claimed in claim 2, wherein the medicament is in an oral dosage form.

14. The combination or use according to claim 13, wherein the medicament is a tablet, capsule, oral liquid, pill, granule or powder.

15. The combination according to claim 1 or the use according to claim 2, wherein the medicament is in an injectable dosage form.

16. The drug combination or the application according to claim 15, wherein the drug is a liquid injection, an oil injection, a milk injection or a powder injection.

17. The combination according to claim 1 or the use according to claim 2, which is used in combination with or comprises a further agent for the treatment of osteosarcoma.

18. The combination or use according to claim 17, wherein the other osteosarcoma treating agent is a chemotherapeutic agent.

19. The combination or use according to claim 17, wherein the other osteosarcoma treating agent is a targeted agent.